Virgin Control Rats Research Articles

The inhibitory effect of dietary fructose on lactation‐induced increases in rat epithelial calcium transport is mediated by fructose‐induced reductions in vitamin D levels

Pregnancy and lactation require marked increases in consumption and transport of dietary Ca2+ to maintain Ca2+ homeostasis and bone health. This increase in epithelial Ca2+ transport is regulated by increasing levels of calcitriol whose genomic effects are mediated by the vitamin D receptor (VDR). We recently showed that fructose (F) consumption reduced intestinal Ca2+ transport. We used lactating rats as animal model to test whether the deleterious effects of excessive F intake on Ca2+ transport is regulated by calcitriol and lead to bone defects. Pregnant and virgin (control) rats were fed isocaloric 63% F or, as controls, glucose and starch diets from d 2 of gestation to end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal Ca2+ transport, elevated intestinal and renal expression of Ca2+ transporters, as well as increased levels of calcitriol and 1‐alpha‐hydroxylase. F consumption prevented these lactation‐induced increases, and reduced VDR binding to promoter regions of TRPV6 and CaBP9k. Bone mineral density, content and mechanical strength each decreased with lactation, and this decrease was exacerbated by F diet. In lactating rats with enhanced Ca2+ needs, excessive F intake prevents, through a calcitriol‐dependent pathway, adaptive increases in epithelial Ca2+ transport, likely compromising bone quality (NSF IOS‐1121049, Benjamin Delessert)

Dietary fructose inhibits lactation‐induced adaptations in rat 1,25‐(OH) 2 D 3 synthesis and calcium transport

We recently showed that excessive fructose consumption, already associated with numerous metabolic abnormalities, reduces rates of intestinal Ca(2+) transport. Using a rat lactation model with increased Ca(2+) requirements, we tested the hypothesis that mechanisms underlying these inhibitory effects of fructose involve reductions in renal synthesis of 1,25-(OH)(2)D(3). Pregnant and virgin (control) rats were fed isocaloric fructose or, as controls, glucose, and starch diets from d 2 of gestation to the end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal transcellular Ca(2+) transport, elevated intestinal and renal expression of Ca(2+) channels, Ca(2+)-binding proteins, and CaATPases, as well as increased levels of 25-(OH)D(3) and 1,25-(OH)(2)D(3). Fructose consumption prevented almost all of these lactation-induced increases, and reduced vitamin D receptor binding to promoter regions of Ca(2+) channels and binding proteins. Changes in 1,25-(OH)(2)D(3) level were tightly correlated with alterations in expression of 1α-hydroxylase but not with levels of parathyroid hormone and of 24-hydroxylase. Bone mineral density, content, and mechanical strength each decreased with lactation, but then fructose exacerbated these effects. When Ca(2+) requirements increase during lactation or similar physiologically challenging conditions, excessive fructose consumption may perturb Ca(2+) homeostasis because of fructose-induced reductions in synthesis of 1,25-(OH)(2)D(3).

CD4 + T-Helper Cells Stimulated in Response to Placental Ischemia Mediate Hypertension During Pregnancy

We have shown that hypertension in response to chronic placental ischemia is associated with elevated inflammatory cytokines and CD4(+) T cells. However, it is unknown whether these cells play an important role in mediating hypertension in response to placental ischemia. Therefore, we hypothesize that reduced uterine perfusion pressure (RUPP)-induced CD4(+) T cells increase blood pressure during pregnancy. To answer this question, CD4(+) T cells were isolated from spleens at day 19 of gestation from control normal pregnant (NP) and pregnant RUPP rats, cultured, and adjusted to 10(6) cells per 100 μL of saline for intraperitoneal injection into NP rats at day 13 of gestation. On day 18, in the experimental groups of rats, arterial catheters were inserted, and on day 19 mean arterial pressure was analyzed. Inflammatory cytokines and antiangiogenic factor soluble fms-like tyrosine kinase 1 were determined via ELISA. Mean arterial pressure increased from 104±2 mm Hg in NP rats to 124±2 mm Hg in RUPP rats (P<0.001) and to 118±1 mm Hg in rats receiving RUPP CD4(+) T cells (P<0.001). Circulating tumor necrosis factor-α and soluble fms-like tyrosine kinase 1 were elevated in recipients of RUPP CD4(+) T cells to levels similar to control RUPP rats. In contrast, virgin rats injected with NP or RUPP CD4(+) T cells exhibited no blood pressure changes compared with control virgin rats. Importantly, mean arterial pressure did not change in recipients of NP CD4(+) T cells (109±3 mm Hg). These data support the hypothesis that RUPP-induced CD4(+) T cells play an important role in the pathophysiology of hypertension in response to placental ischemia.

MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic β-cell proliferation in islets from early lactating mothers

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phospho-ERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.

Mechanisms Underlying Maternal Venous Adaptation in Pregnancy

To define the effects of pregnancy on mechanical properties and reactivity, mesenteric veins from late pregnant and virgin control (nonpregnant) rats were pressurized to determine gestational changes in size and distensibility. Reactivity studies used an adrenergic constrictor (norepinephrine) and an endothelium-mediated vasodilator (acetylcholine). The contribution of nitric oxide to endothelial function was evaluated with pharmacologic inhibition of nitric oxide synthase. Roles of nitric oxide and cyclic guanosine monophosphate in smooth muscle vasodilation were determined using an nitric oxide donor with and without cyclic guanosine monophosphate inhibition using ODQ, a selective inhibitor of guanylyl cyclase. In pregnancy, endothelium-dependent vasodilation markedly increased (largely due to endogenous nitric oxide), smooth muscle response to nitric oxide decreased (primarily related to cyclic guanosine monophosphate production), and norepinephrine sensitivity decreased considerably, with no changes in vessel size or distensibility. Our results identify a provasodilatory state in the systemic venous system, which would serve to facilitate the accommodation to plasma volume expansion requisite for normal pregnancy.

Pregnancy and dexamethasone: Effects on morphometric parameters of gonadotropic cells in rats

The effects of pregnancy and multiple dexamethasone (Dx) treatment on morphometric parameters of adenohypophyseal gonadotropic cells that produce follicle stimulating hormone (FSH) and luteinizing hormone (LH) were investigated in female Wistar rats. The rats in the experimental group received injections of 1.0, 0.5 and 0.5mg Dx/kg b.w. on days 16-18 of pregnancy, while the control group received equal volumes of saline. There was also an age-matched adult virgin control group. The experimental and control animals were sacrificed 24 and 72h after the last injection. Using the peroxidase-anti-peroxidase immunohistochemical labeling procedure, morphometric analyses showed that cell volume and volume density of FSH and LH cells on day 19 of pregnancy, as well as the number of LH cells, were significantly decreased compared to the virgin control values. On day 21 of gestation, the volume of FSH and LH cells remained smaller than in the virgin controls. Moreover, FSH and LH cell volume was significantly decreased 24h after multiple Dx treatment in comparison with the pregnant controls. Thus, during the last days of pregnancy, the morphometric parameters of gonadotropic cells decreased in comparison with the control virgin rats, but Dx treatment of pregnant rats had an inhibitory influence on FSH and LH cell size only 24h after the last dose.

The Hypo-Osmolality of Pregnancy Does Not Alter Baroreceptor Responses to Acute Changes in Osmolality

Reducing extracellular osmolality by 10-12 mOsm/L reduces baroreceptor firing in vitro in tissues from male rats. Pregnancy is associated with a similar reduction in plasma osmolality (pOsm) as well as with alterations in baroreceptor firing and reduced baroreceptor reflex activity. Mechanisms for reduced baroreflex activity are not yet understood, but they have important implications for maintenance of cardiovascular homeostasis in the pregnant female. Thus, this study was designed to test whether changes in plasma osmolality alter baroreceptor discharge during pregnancy. Late-gestation pregnant and virgin control rats were anesthetized, femoral vessels were cannulated for measurement of arterial pressure and drug infusion, and the aortic depressor nerve, containing baroreceptor afferents, was isolated and prepared for recording. Plasma osmolality was measured before and 30 min after graded intraperitoneal injections of NaCl (50-1500 mOsm/L). Arterial pressure, heart rate, and aortic depressor nerve activity (ADNA) were measured continuously before and after injections. A 50 mOsm/L NaCl injection significantly decreased pOsm but did not alter ADNA in either group. Likewise, 1200 mOsm/L NaCl injections significantly increased pOsm and had no effect on ADNA in either group. The 1500 mOsm salt load significantly increased pOsm and ADNA in pregnant rats, and in virgin animals, it increased pOsm but evoked a paradoxical decrease in ADNA. The authors' studies indicate that pOsm has minimal effects on baroreceptor activity, which are not significantly different during gestation. Their findings suggest that in vitro data from male rats indicating a correlation between pOsm and baroreceptor discharge are not applicable in anesthetized intact female animals.

Changes in cytochrome P450 isozymes (CYPs) protein levels during lactation in rat liver

The effects of pregnancy on CYPs protein level in the liver have been investigated in our previous study. Since pregnancy was associated with a decrease in CYPs protein level, the objective of this study was to investigate whether CYPs protein can revert to the virgin control level after delivery. Western blot analysis was performed to investigate the changes of total nine CYPs protein (CYP1A1, CYP2B1/CYP2B2, CYP2C6, CYP2C12, CYP2D1, CYP2D4, CYP2E1, CYP3A1 and CYP4A1) at three distinct phases: delivery (postpartum day 0, PPD 0), peak lactation (PPD 14) and on weaning (PPD 28). By PPD 0, CYP1A1, 2B1, 2B2, 2C6, 2E1 and CYP4A1 were markedly down-regulated when compared with virgin controls. By PPD 14, however, CYP1A1, 2B1, 2B2 and CYP2C6 returned to the virgin control level. All the decreased CYPs during lactation were at the virgin control level at PPD 28. The expression of CYP2C12, CYP2D1 and CYP 3A1 did not differ between lactating, post-lactation and virgin control rats. CYP2D4 was not detectable in microsomal proteins obtained from virgin control rats at a protein loading of 20 μg total protein per lane.

Elevated Expression of Liver γ-Cystathionase Is Required for the Maintenance of Lactation in Rats

Liver γ-cystathionase activity increases in rats during lactation; its inhibition due to propargylglycine is followed by a significant decrease in lactation. This is reversible by N-acetylcysteine administration. To study the role of liver γ-cystathionase and the intertissue flux of glutathione during lactation, we used lactating and virgin rats fed liquid diets. Virgin rats were divided into two groups as follows: one group was fed daily a diet containing the same amount of protein that was consumed the previous day by lactating rats (high protein diet–fed rats); the other virgin group was fed the normal liquid diet (control). The expression and activity of liver γ-cystathionase were significantly greater in lactating rats and in high protein diet–fed virgin rats compared with control rats. The total glutathione [reduced glutathione (GSH) + oxidized glutathione (GSSG)] released per gram of liver did not differ in lactating rats or in high protein diet–fed rats, but it was significantly higher in these two groups than in control virgin rats. Liver size and the GSH + GSSG released by total liver were significantly higher in lactating rats than in high protein diet–fed virgin rats, and this difference was similar to the amount of glutathione taken up by the mammary gland (454.2 ± 36.0 nmol/min). The uptake of total glutathione by the lactating mammary gland was much higher than the uptakes of free L-cysteine and L-cystine, which were negligible. These data suggest that the intertissue flux of glutathione is an important mechanism of L-cysteine delivery to the lactating mammary gland, which lacks γ-cystathionase activity. This emphasizes the physiologic importance of the increased expression and activity of liver γ-cystathionase during lactation.

Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes.

1. The responsiveness of the rat hypothalamo-pituitary-adrenal (HPA) axis and hypothalamo-neurohypophysial system (HNS) to emotional (elevated plus-maze) and physical (forced swimming) stressors and to administration of synthetic corticotrophin-releasing hormone (CRH) was investigated during pregnancy and lactation. In addition to pregnancy-related adaptations at the adenohypophysial level, behavioural responses accompanying the neuroendocrine changes were studied. 2. Whereas basal (a.m.) plasma corticosterone, but not corticotrophin (adrenocorticotrophic hormone; ACTH), levels were increased on the last day (i.e. on day 22) of pregnancy, the stress-induced rise in both plasma hormone concentrations was increasingly attenuated with the progression of pregnancy beginning on day 15 and reaching a minimum on day 21 compared with virgin control rats. A similar attenuation of responses to both emotional and physical stressors was found in lactating rats. 3. Although the basal plasma oxytocin concentration was elevated in late pregnancy, the stress-induced rise in oxytocin secretion was slightly lower in day 21 pregnant rats. In contrast to vasopressin, oxytocin secretion was increased by forced swimming in virgin and early pregnant rats indicating a differential stress response of these neurohypophysial hormones. 4. The blunted HPA response to stressful stimuli is partly due to alterations at the level of corticotrophs in the adenohypophysis, as ACTH secretion in response to CRH in vivo (40 ng kg-1, i.v.) was reduced with the progression of pregnancy and during lactation. In vitro measurement of cAMP levels in pituitary segments demonstrated reduced basal levels of cAMP and a lower increase after CRH stimulation (10 nM, 10 min) in day 21 pregnant compared with virgin rats, further indicating reduced corticotroph responsiveness to CRH in pregnancy. 5. The reduced pituitary response to CRH in late pregnancy is likely to be a consequence of a reduction in CRH receptor binding as revealed by receptor autoradiography. [125I] CRH binding in the anterior pituitary was significantly reduced in day 11, 17 and 22 pregnant rats compared with virgin controls. 6. Anxiety-related behaviour of the animals as revealed by the time on and entries into the open arms of the elevated plus-maze was different between virgin and pregnant rats with decreased number of entries indicating increased anxiety with the progression of pregnancy (except on pregnancy day 18). The emotional behaviour, however, was not correlated with the neuroendocrine responses. 7. The results indicate that the reduced response of the HPA axis to stressors described previously during lactation is already manifested around day 15 of pregnancy in the rat and involves physiological adaptations at the adenohypophysial level. However, alterations in stressor perception at higher brain levels with the progression of pregnancy may also be involved.

Long-term nitric oxide blockade in the pregnant rat: Effects on blood pressure and plasma levels of endothelin-1

OBJECTIVE: Abnormalities in the production of nitric oxide and endothelin-1 have been implicated in the development of preeclampsia. We postulated that long-term nitric oxide synthase inhibition with L-nitro-arginine methyl ester would induce sustained hypertension, a rise in plasma levels of endothelin-1, and fetal growth restriction. STUDY DESIGN: Conscious virgin and pregnant Sprague-Dawley rats received infusions of vehicle or L-nitro-arginine methyl ester (2.5 mg/kg/hr) for 11 days. Mean arterial pressure was assessed serially. On day 21 of gestation (or equivalent in virgin rats) plasma was collected for endothelin-1 levels; pup weight and litter size were determined. Data were analyzed with analysis of variance and regression techniques. RESULTS: Mean arterial pressure was constant in virgin control rats ( n = 7) but declined in pregnant control rats ( n = 11) as gestation advanced. Nitric oxide synthase inhibition in virgin ( n = 10) and pregnant ( n = 11) rats caused sustained elevations in mean arterial pressure (165 ± 7 vs 100 ± 3 mm Hg, L-nitro-arginine methyl ester vs control virgin rats, p < 0.0001; 149 ± 5 vs 91 ± 2 mm Hg, L-nitro-arginine methyl ester vs control pregnant rats, p < 0.0001). L-nitro-arginine methyl ester induced a rise in plasma endothelin-1 levels in virgin (4.4 ± 0.1 vs 3.5 ± 0.1 pg/ml, L-nitro-arginine methyl ester vs control, p < 0.0001) and pregnant rats (3.0 ± 0.1 vs 2.6 ± 0.1 pg/ml, L-nitro-arginine methyl ester vs control, p < 0.0001). Pregnant rats had lower endothelin-1 levels than did virgin rats ( p < 0.0001). Mean arterial pressure and endothelin-1 were significantly correlated in pregnant rats. L-nitro-arginine methyl ester decreased pup weight (2.4 ± 0.4 vs 3.7 ± 0.2 gm/pup/litter, L-nitro-arginine methyl ester vs control, p < 0.01) and litter size (6.6 ± 1.3 vs 10.2 ± 0.9 pups/litter, L-nitro-arginine methyl ester vs control, p < 0.05). CONCLUSIONS: Long-term nitric oxide synthase blockade causes sustained hypertension, elevated levels of endothelin-1, and fetal growth restriction. Although the endocrine and pressor effects are not unique to pregnancy, this model clearly induces some of the changes seen in preeclampsia and may be useful for studying specific interventions. (Am J Obstet Gynecol 1996;175:484-8.)

The set point for maternal glucose homeostasis is lowered during late pregnancy in the rat: the role of the islet beta-cell and liver.

The aim of this study was to determine the effects of late pregnancy on the ability of insulin to suppress maternal hepatic glucose production in the rat. Unlike in most previous studies, suppression of hepatic glucose production was measured at levels of glycaemia above the relatively hypoglycaemic basal pregnant level. Glucose kinetics were measured using steady-state tracer methodology in chronically catheterised, conscious virgin control and pregnant rats, firstly, during basal and low-dose hyperinsulinaemic euglycaemic clamp conditions and secondly, during a three-step glucose infusion protocol (glucose infusion rates of 0, 60 and 150 mumol.kg-1. min-1). During the clamps, plasma glucose levels were not different (6.1 +/- 0.4 vs 6.5 +/- 0.3 mmol/l, pregnant vs virgin; N.S.), but plasma insulin levels were higher in the pregnant rats (242 +/- 30 vs 154 +/- 18 pmol/l. pregnant vs virgin; p < 0.05) most probably due to stimulated endogenous insulin release in this group. Hepatic glucose production was suppressed from basal levels by 41% in virgin and 90% in pregnant rats. During the glucose infusion studies, at matched insulin levels (147 +/- 10 vs 152 +/- 14 pmol/l), but at plasma glucose levels which were much lower in the pregnant rats (5.5 +/- 0.2 vs 8.4 +/- 0.6 mmol/l, pregnant vs virgin; p < 0.0001), hepatic glucose production was shown to be suppressed by a similar degree in both groups (41 +/- 5 vs 51 +/- 5% from basal, pregnant vs virgin; N.S.). Both the plasma insulin and percentage suppression of hepatic glucose production dose responses to plasma glucose were markedly shifted to the left indicating that the plasma glucose set point is lowered in pregnancy. In conclusion, suppression of hepatic glucose production by insulin is not impaired and the set point for plasma glucose homeostasis is lowered during late pregnancy in the rat.

Effect of diabetes on protein synthesis rate and eukaryotic initiation factor activities in the liver of virgin and pregnant rats.

To study the effect of prolonged diabetes on protein synthesis and on the activities of initiation factors eIF-2 and eIF-2B in the liver, female rats were treated with streptozotocin. Some animals were mated and studied on day 20 of pregnancy, whereas others were kept virgin and studied in parallel. The protein synthesis rate was measured with an "in vitro' cellfree system, and was lower in diabetic pregnant and virgin animals than in pregnant and virgin controls (30-60%). The fetuses of diabetic rats had a lower protein synthesis rate than those from controls, although they always showed a higher protein synthesis rate than their mothers or virgin rats. Protein synthesis rate, RNA concentration, and initiation factor 2 activity were higher in pregnant than in virgin rats. Both activity and level of eIF-2 factor changed in parallel to the protein synthesis rate, although no differences could be detected between control and diabetic animals. The eIF-2B activity in tissue extracts from diabetic virgin rats and fetuses was lower than in extracts from their controls, whereas no differences could be detected between pregnant and virgin control rats nor between pregnant control and pregnant diabetic animals. The percentage of the phosphorylated form of eIF-2 factor, eIF-2(alpha P), was slightly lower in virgin than in pregnant rats but was unaffected by the diabetic condition, while in diabetic fetuses this parameter was lower than in their corresponding controls. The cyclic adenosine monophosphate dependent protein kinase level was lower in diabetic rats than in controls, whereas no changes in the activity of casein kinase II were found. The isoelectric forms of the beta subunit of eIF-2 factor, eIF-2 beta, were different in the diabetic and the control animals, indicating that insulin deficiency modifies the phosphorylation of specific substrates. Since no differences were detected in RNA or eIF-2 content between control and diabetic rats, translation may, at least partly, be inhibited in the liver by an impairment of peptide chain initiation caused by the decreased eIF-2B activity which nevertheless is independent of eIF-2 alpha phosphorylation.

Reversion of insulin resistance in the rat during late pregnancy by 72-h glucose infusion.

To determine whether sustained exaggerated hyperinsulinemia in normoglycemic rats modifies insulin responsiveness during pregnancy, 17-day-pregnant and virgin rats were studied after receiving a continuous intravenous infusion (35 ml/day) of either 50% glucose or bidistilled water (controls) for 72 h. Plasma glucose was unchanged, whereas insulin was highly increased, and the effect was more marked in pregnant than in virgin rats. Insulin responsiveness, estimated under the hyperinsulinemic euglycemic clamp with 0.8 IU insulin.h-1.kg-1, was lower in control pregnant than in virgin rats but higher in pregnant than in virgin rats in those that had received the glucose infusion. The tissue glucose utilization metabolic index (GUI) was estimated with 2-deoxy-D-[1-3H]glucose in the clamped rats. The GUI was lower in heart, white- and red-fiber skeletal muscle, and adipose tissue in control pregnant rats than in control virgin rats, and, although the glucose infusion decreased that index in both red-fiber muscle and adipose tissue in virgin rats, glucose increased the index in red-fiber muscle in pregnant rats to the level found in virgin controls. Results therefore show that, when unaccompanied by hypoglycemia, sustained exaggerated hyperinsulinemia decreases insulin responsiveness in virgin rats but reverts insulin resistance in late-pregnant rats.

Expression of rat microsomal epoxide hydrolase during pregnancy

Microsomal epoxide hydrolase (mEH) protein and messenger ribonucleic acid (mRNA) levels were assessed in maternal rats during pregnancy and mEH gene expression was compared with cytochrome P450 expression. Immunoblot analysis using goat anti-rat mEH antibody showed that hepatic mEH levels in adult rats at day 12 of gestation were comparable to those in virgin female rats at the same age, whereas mEH protein levels were substantially decreased by ~70% at day 20 of pregnancy. Northern and slot blot analyses revealed that hepatic mEH mRNA levels failed to be modulated at day 12 of pregnancy, whereas mEH mRNA levels were decreased to ~20% of virgin control in late pregnancy ( i.e. day 20), which was consistent with the result of mEH immunoblot analysis. Hepatic cytochrome P4502E1 levels were also diminished at day 12 and day 20 of gestation by ~50% and ~70%, respectively, relative to virgin control rats, as supported by immunoblot analysis. Hepatic P4502E1 mRNA levels at day 12 and day 20 of pregnancy were also significantly decreased to 30% and 8% of virgin rats at 17 weeks of age, respectively, demonstrating that suppression in P4502E1 protein levels accompanied decreases in its mRNA expression. The levels of cytochrome P4501A2 mRNA at either day 12 or day 20 of pregnancy was decreased by ~50% relative to virgin female rats, which was less than that observed in mEH or P4502E1 expression. Pregnancy failed to affect P4501A1 mRNA levels, which were not detectable in female rats at 17 weeks of age. Renal mEH mRNA levels in maternal rats at day 20 of gestation were also decreased to 25% of virgin control. These results demonstrated that levels of mEH and P4502E1 proteins decreased significantly in late pregnancy with concomitant decreases in their mRNA levels.

Lactation as a model for naturally reversible hypercorticalism plasticity in the mechanisms governing hypothalamo-pituitary- adrenocortical activity in rats.

Steady state levels of hypothalamic expression of the genes encoding corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), arginine vasopressin (AVP), and oxytocin (OT) were studied in rats to investigate the mechanisms underlying the transitions between hypercorticalism during lactation and normocorticalism upon weaning. During lactation, CRH mRNA levels and blood titers of adrenocorticotropin (ACTH) were found to be significantly reduced, although POMC mRNA levels in the anterior pituitary were not significantly different from those found in cycling virgin (control) rats; during all phases of lactation, an inverse relationship was observed between the blood levels of ACTH and corticosterone (CORT). Plasma prolactin (PRL) concentrations were elevated approximately 30-fold during lactation. Whereas steady state levels of OT mRNA were markedly increased throughout lactation, those of AVP mRNA were only transiently (initially) elevated, and the blood levels of these hormones were not significantly altered in lactating as compared with cycling virgin and postlactating rats. CRH and POMC gene expression and blood levels of ACTH, CORT, and PRL were normalized within 1-3 d of removal of suckling pups. The temporal relationships between the biosynthetic profiles of the various peptide hormones and the patterns of ACTH and CORT secretion during the two physiological states suggest that lactation-associated hypercorticalism does not merely result from increased ACTH secretion; although still not well substantiated at this time, the evidence points to contributory roles of PRL, OT, and AVP in the hypercorticalismic state found during lactation.

Effect of streptozotocin diabetes on polysomal aggregation and protein synthesis rate in the liver of pregnant rats and their offspring.

To study the effect of diabetes on hepatic protein synthesis and polysomal aggregation in pregnant rats, female rats were treated with streptozotocin prior to conception. Some animals were mated, and studied at day 20 of pregnancy, whereas, others were studied in parallel under non pregnant conditions. The protein synthesis rate measured with an "in vitro" cell-free system was higher in pregnant than in virgin control rats. It decreased with diabetes in both groups, although values remained higher in diabetic pregnant rats than in the virgin animals. The fetuses of diabetic rats had a lower protein synthesis rate than those from controls, although they showed a higher protein synthesis rate than either their respective mothers or virgin rats. Liver RNA concentration was higher in control and diabetic, pregnant rats than in virgin rats, and the effect of diabetes decreasing this parameter was only significant for pregnant rats. Liver RNA concentration in fetuses was lower than in their mothers, and did not differ between control and diabetic animals. The decreased protein synthesis found in diabetic animals was accompanied by disaggregation of heavy polysomes into lighter species, indicating an impairment in peptide-chain initiation.

Glucose and Insulin Tolerance Tests in the Rat on Different Days of Gestation

To study insulin/glucose relationship during gestation, rats were studied on days 6, 12, 15, 18, 20 or 21 of pregnancy and the results were compared to values in sex-matched virgin control rats. Blood glucose levels were decreased on days 20 and 21 of gestation whereas plasma insulin levels appeared decreased on days 6 and 12, unchanged on day 15 and enhanced on days 18, 20 and 21 of gestation. Total pancreas insulin content was already augmented on day 6 of gestation and continued to increase with gestational time. With the exception of an increase in the 6-day-pregnant rats 22.5 min after an oral glucose load, blood glucose levels did not differ between 6- or 12-day-pregnant rats and virgin controls although plasma insulin levels reached higher values on these days. However, in the 15-day-pregnant rats, glucose tolerance after the glucose load was enhanced while plasma insulin levels did not differ from those in virgin rats during the first 30 min. In the 18-day-pregnant rat blood glucose was more increased but plasma insulin did not differ after the glucose load when compared to virgin rats, whereas 20- or 21-day-pregnant rats showed a glucose tolerance similar to that of virgin rats but their insulin levels shortly after the glucose load were higher. The hypoglycemic response to a high intravenous dose of insulin was decreased in 12-, 18-, 20- and 21-day-pregnant rats. Therefore, whereas in both the 6- and 12-day-pregnant rats there is an enhanced beta-cell response to the glucose insulinotropic effect and insulin responsiveness is reduced in 12-day-pregnant rats, the 15-day pregnant rat is in a transitory stage where both insulin sensitivity and the beta-cell response return to nonpregnant values. However, from 18 days of gestation on, there is an intense insulin-resistant condition which is only partially compensated by an enormous accumulation of insulin in the pancreas followed by a faster and larger insulin release after a glucose load.

Hepatic Amino Acid Uptake Is Decreased in Lactating Rats. In Vivo and In Vitro Studies

To study the redistribution of amino acids to the mammary gland during lactation we used lactating and virgin rats fed liquid diets. Virgin rats were divided in two groups: one group was fed daily a diet containing the same amount of protein that was consumed the previous day by lactating rats (high protein diet-fed rats), and the other virgin group was fed the normal liquid diet (control). The hepatic availability of amino acids was significantly higher in the lactating rats than in the other two groups, but the uptake and fractional extraction of amino acids by the liver were lower in lactating rats than in the high protein-fed virgin controls. When primary hepatocyte cultures were used, the uptake of 2-amino-[1-14C]isobutyric acid (AIB) and the activity of system A were found to be significantly higher in the hepatocytes from virgin rats fed the high protein diet than in those obtained from the lactating and control virgin groups. No difference was observed between the control virgin rats and the lactating rats. The kinetic of AIB showed that the Vmax/Km ratio was significantly lower in hepatocytes from lactating rats than in those from the high protein diet-fed virgin rats. Addition of prolactin to the incubation medium decreased the uptake of AIB in hepatocytes from both groups of virgin rats. Moreover, uptake of AIB was greater in bromocriptine-treated lactating rats and in lactating rats that had had their pups removed for the preceding 24 h compared with values for the lactating rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein lipase and hormone-sensitive lipase activity and mRNA in rat adipose tissue during pregnancy.

To investigate the factors controlling maternal depot fat accumulation during early pregnancy and net decrease during late pregnancy, the activity and mRNA expression of adipose tissue lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) were related to several other lipid metabolic parameters. Virgin control rats, pregnant rats (at days 12, 15, 19, and 21), and lactating rats (at days 5 and 10 postpartum) were studied. In adipose lumbar tissue of late pregnant rats, LPL activity decreased to about one-third that of the virgin control animals, with < 10% of initial LPL mRNA expressed as determined by Northern blots. HSL activity increased maximally 1.5-fold with a fourfold increase of HSL expression at days 12-15 of pregnancy and decreased to control levels after parturition. The HSL-to-LPL mRNA and activity ratios were enhanced from days 15 and 19 of pregnancy, respectively, and remained so even during lactation, mainly because of the marked lowering of the LPL values. This enhancement coincided with increments in plasma free fatty acids and glycerol levels indicating an increased depot fat breakdown. These results give no indication of an involvement of LPL and HSL gene expression changes in the accumulation of maternal depot during early pregnancy. In contrast, such changes could be responsible for the net breakdown of this fat depot during late gestation. Thus, during this physiological state, long-term (e.g., transcriptional) regulation of LPL and HSL gene expression could be an important mechanism for the control of adipose tissue mass breakdown during late gestation.